1. Field of the Invention
The present invention provides antisense oligodeoxynucleotides targeted to exon sequences flanking donor splice sites which regulate expression of TNF-xcex1.
2. Description of Related Art
There has been increasing interest in the development of antisense oligodeoxyribonucleotides (ASxe2x80x94ODNs hereinafter) as therapeutic agents and experimental tools (Stein and Cheng, 1993; Wagner, 1994). However, despite the improvement in affinity for target RNA, increased resistance to nucleolytic cleavage, and enhanced delivery of ASxe2x80x94ODNs to cells and their nuclei (Hodges and Crooke, 1995), high concentrations of ASxe2x80x94ODNs continue to be required to inhibit gene expression. To some extent, high ASxe2x80x94ODN concentrations have hampered the development of this technology as an effective pharmacological agent because of cost and non-specific AS-ODN actions.
Many genes encode pre-mRNAs containing introns that are removed by a splicing process that is directed by a complex of small nuclear ribonucleic proteins (snRNPs) called the spliceosome (Staley and Guthrie, 1998). Several reports indicate that gene expression is effectively inhibited by ASxe2x80x94ODNs targeting the intron/exon boundaries of splice sites (Boeve and De Ley, 1994; Dominski and Kole, 1996; Dominski and Kole, 1994; Hodges and Crooke, 1995; Moulds et al., 1995), likely because these domains direct splicing events (Staley and Guthrie, 1998). It has previously been shown in cell free systems that the degree of sequence variability at splice sites influences splicing events (Dominski and Kole, 1994), suggesting that pre-mRNAs with variant splice site sequences would be ideal targets for ASxe2x80x94ODN treatment (Hodges and Crooke, 1995). Since exon sequences upstream of donor (5xe2x80x2), and downstream of acceptor (3xe2x80x2) splice sites within pre-mRNA play a critical role in processing RNA (Staley and Guthrie, 1998), it is plausible that these sites encode RNA domains highly susceptible to ASxe2x80x94ODN-mediated inhibition of gene expression. To date, this hypothesis has not been tested systematically in a biologically relevant system such as tumor necrosis factor alpha (TNF-xcex1) production in cell culture or in vivo.
Under normal conditions, TNF-xcex1""s expression is tightly regulated by rapid mRNA turnover (Gearing et al., 1995). However, in disease states, its expression is perturbed, resulting in overexpression (Sharief and Hentges, 1991; Tracey and Cerami, 1994). TNF-xcex1 is implicated in the pathogenesis of several inflammatory diseases including multiple sclerosis (MS) (French-Constant, 1994), rheumatoid arthritis (RA) (Lupia et al., 1996), viral infections such as human immunodeficiency virus (HIV) (Fauci, 1996) and, bacterial infections causing sepsis (Tomioka et al., 1996). TNF-xcex1 neutralizing antibodies (Givner et al., 1995), soluble TNF-xcex1 receptors (Moreland et al., 1997), or gene knockouts of the TNF receptor (p55) (Pfeffer et al., 1993) mitigate the harmful effects of TNF-xcex1 observed in several animal models of inflammation (Probert et al., 1995; Selmaj et al., 1991). However, these approaches do not limit TNF-xcex1 synthesis.
Several studies show that ASxe2x80x94ODNs targeting TNF-xcex1 mRNA limit TNF-xcex1 synthesis (Hartmann et al., 1996; Lefebvre d""Hellencourt et al., 1996; Rojanasakul et al., 1997; Taylor et al., 1996). However, in these reports, concentrations of ASxe2x80x94ODNs in excess of 2 xcexcM, were required to achieve significant inhibition. High concentrations of ASxe2x80x94ODNs may induce non-specific inflammatory cell responses (Hartmann et al., 1996) as well as other non-specific effects (Gao et al., 1992; Khaled et al., 1996; Perez et al., 1994). Nevertheless, earlier reports suggest that expression of other genes can be regulated by low concentrations (xe2x89xa61 xcexcM) of ASxe2x80x94ODNs (Hanecak et al., 1996; Miraglia et al., 1996). Therefore it would be useful to develop ASxe2x80x94ODNs that can be used in low concentrations to regulate TNF-xcex1 production in inflammatory responses.
According to the present invention, a synthetic nuclease resistant antisense oligodeoxynucleotide capable of selectively modulating expression of human tumor necrosis factor-alpha by targeting exon sequences flanking donor splice sites, thereby regulating expression of TNF-xcex1 in a patient in need of such therapy is provided. In an embodiment either ASxe2x80x94ODN having the sequence set forth in SEQ ID No:4 or SEQ ID No:6 or a combination thereof can be used. The ASxe2x80x94ODN is administered either as the active ingredient in a pharmaceutical composition or by utilizing gene therapy techniques as an expression vector.